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FCV rescue system establishment and GFP expression
Author: LiWuXin
Tutor: WangGuiPing
School: Jilin University
Course: Preventive Veterinary Medicine
Keywords: Feline calicivirus Virus rescue GFP
CLC: S852.5
Type: Master's thesis
Year: 2008
Downloads: 55
Quote: 0
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Abstract
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For FCV CH-GD strains of the whole gene cloning and sequencing . The virus has a typical calicivirus structure , its genome is a single strand containing the polyA RNA. Length of 7756 bp, encoding three open reading frames (ORFs). In this study, the RT-PCR method to obtain the FCV CH-GD strain genome cDNA. The PCR method FCV 5 ' end of the introduction of hammerhead ribozyme (Hammerhead ribozyme) cDNA sequence, the virus full-length cDNA fragment was cloned into the expression plasmid pcDNA-HdvRz , the recombinant plasmid pFCV-HdvRz. The recombinant plasmid pFCV-HdvRz F81 cells transfected successfully constructed FCV infectious clone . Through cell disease, indirect immunofluorescence reaction , RT-PCR and other methods to identify the rescued viruses . The results show that: ( 1 ) the cells can be observed lesions, viral titer no difference compared with the original drug ; ( 2 ) in the indirect immunofluorescence test can be observed in the cytoplasm -specific fluorescence , while the negative serum control group fluorescence can not be observed . ( 3 ) the cell culture extract total RNA, RT-PCR was amplified and sequenced to obtain the desired fragment . On this basis , the green fluorescent protein gene into the FCV capsid protein gene back and connected at both ends to insert FCV 3C -like protease hydrolysis site cDNA sequences to construct expressing green fluorescent protein infectious clone carrier. F81 cells transfected , green fluorescence was observed . Determined by observing GFP fluorescence in the green FCV whole genome expression , expression of the other so as to lay the foundation for the foreign gene .
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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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